Method for preparing amg 416 (etelcalcetide)

ABSTRACT

A method for preparing AMG 416, or a pharmaceutically acceptable salt thereof, is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 16/532,344, filed Aug. 5, 2019, now allowed, which is acontinuation of U.S. Non-Provisional application Ser. No. 15/300,209,filed Sep. 28, 2016, now issued as U.S. Pat. No. 10,407,464, which is aU.S. National Stage Application of International Patent Application No.PCT/US2015/024347, filed Apr. 3, 2015, which claims the benefit of U.S.Provisional Application No. 61/974,899, filed Apr. 3, 2014, each ofwhich is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

A sequence listing is being submitted electronically via EFS in the formof a text file, created Jun. 28, 2022, and named “0419251014seqlist.txt”(2730 bytes), the contents of which are incorporated herein by referencein their entirety.

FIELD

The present disclosure relates to the field of polypeptide synthesis,and more particularly to the synthesis of AMG 416, or a pharmaceuticallyacceptable salt thereof.

BACKGROUND

AMG 416 is a synthetic, eight amino-acid selective peptide agonist ofthe calcium sensing receptor. It is being developed as an intravenoustreatment of secondary hyperparathyroidism (SHPT) in hemodialysispatients with chronic kidney disease-mineral and bone disorder(CKD-MBD).

The hydrochloride salt of AMG 416 has the chemical structure:

The main chain has 7 amino acids, all in the D-configuration. Theside-chain cysteine residue is in the L-configuration. The molecularformula of AMG 416 (free base) is C₃₈H₇₃N₂₁O₁₀S₂, and has a calculatedaverage molecular mass of 1048.3 Da.

AMG 416 and a method for its preparation are described in InternationalPat. Publication No. WO 2011/014707, which is incorporated herein byreference for any purpose. As described in International Pat.Publication No. WO 2011/014707, AMG 416 may be assembled by solid-phasesynthesis from the corresponding Fmoc-protected D-amino acids. Aftercleavage from the resin, the material may be treated withBoc-L-Cys(NPyS)—OH to form the disulfide bond. The Boc group may then beremoved with trifluoroacetate (TFA) and the resulting product purifiedby reverse-phase high pressure liquid chromatography (HPLC) and isolatedas the TFA salt form by lyophilization. The TFA salt can be converted toa pharmaceutically acceptable salt by carrying out a subsequent saltexchange procedure. Such procedures are well known in the art andinclude, e.g., an ion exchange technique, optionally followed bypurification of the resultant product (for example by reverse phaseliquid chromatography or reverse osmosis).

There is a need for an efficient method of producing AMG 416, or apharmaceutically acceptable salt thereof (e.g., AMG 416 HCl), andparticularly one appropriate for commercial scale manufacturing.

SUMMARY

In view of the above-described problems, it is an objective of thedisclosure to provide a method for preparing AMG 416, or apharmaceutically acceptable salt thereof, among other things.

In a first aspect, provided is a method for preparing AMG 416, themethod comprising: providing a resin-bound peptide having a structureselected from the group consisting ofFmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin](SEQ ID NO:2) andAc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin](SEQ ID NO:3); cleaving the peptide from the solid support; andactivating the side chain of the D-Cys residue of the cleaved peptide.

In one or more embodiments related to the first aspect, the cleaving andthe activating steps occur in the same vessel.

In one or more further embodiments, the resin-bound peptide is contactedwith a solution comprising water, trifluoroacetic acid,triisopropylsilane and dipyridyldisulfide.

In a second aspect, provided is a method for preparing AMG 416, themethod comprising: providing a peptide having a structure ofAc-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂ (SEQ ID NO:4); andcontacting the peptide with L-Cys to produce a conjugated product.

In some embodiments related to the second aspect, the peptide iscontacted with an aqueous solution comprising L-Cys and trifluoroaceticacid.

In some further embodiment related to the second aspect, the methodfurther comprises lyophilizing the conjugated product.

In yet some further embodiments, the method of the second aspect furthercomprises contacting the conjugated product with an aqueous solutioncomprising isopropyl alcohol (IPA) and hydrochloric acid (HCl), therebyproducing a precipitate comprising AMG 416 HCl.

In yet one or more further embodiments related to the second aspect, themethod further comprises purifying the precipitate by high performanceliquid chromatography (HPLC).

In yet a third aspect provided is a method for preparing AMG 416, themethod comprising: providing a resin-bound peptide having a structureselected from the group consisting ofFmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin](SEQ ID NO:2) andAc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin](SEQ ID NO:3); cleaving the peptide from the solid support, i.e., toprovide an unsupported peptide, and activating the side chain of theD-Cys residue of the unsupported peptide to generate an AMG 416 SPyintermediate (where SPy is 2-pyridinesulfenyl or S-Pyr), dissolving theAMG 416 SPy intermediate in an aqueous 0.1% TFA (trifluoroacetic acidsolution), and purifying the AMG 416 SPy derivative by HPLC.

In some embodiments related to the third aspect, the method furthercomprises azeotropic distillation of the AMG 416 SPy intermediate, tothereby effect a solvent exchange to produce a solution of the AMG 416SPy in the new solvent, e.g., water and isopropyl alcohol.

In yet some additional embodiments related to the third aspect, themethod further comprises contacting the isopropyl alcohol-water solutionof the AMG 416 SPy, in some embodiments in the form of itstrifluoroacetate salt, with an aqueous solution comprising L-Cys.

In a fourth aspect, provided is a method for preparing H-D-Arg(Pbf)-OH,i.e., a suitable starting material for certain of the synthetic methodsprovided herein.

In some embodiments related to the fourth aspect, the method comprisesconverting Boc-D-Arg-OH to Boc-D-Arg(Pbf)-OH in the presence of NaI(sodium iodide). In yet some further embodiments, the sodium iodide ispresent at a concentration of about 5% mol.

In some additional embodiments related to the fourth aspect, the methodfurther comprises converting Boc-D-Arg(Pbf)-OH to D-Arg(Pbf)-OH, andcrystallizing the D-Arg(Pbf)-OH in an IPA/water solvent system.

Additional embodiments of the methods described herein will be apparentfrom the following description, examples, and claims. As can beappreciated from the foregoing and following description, each and everyfeature described herein, and each and every combination of two or moreof such features, is included within the scope of the present disclosureprovided that the features included in such a combination are notmutually inconsistent. In addition, any feature or combination offeatures may be specifically excluded from any embodiment of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of AMG 416(Ac-D-Cys(L-Cys-OH)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂) (SEQ IDNO:1).

FIG. 2 shows the chemical structure of Rink Amide AM resin andAc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-Resin(SEQ ID NO:3).

FIG. 3 shows a reaction scheme in which the SPy intermediate product(Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂) (SEQ ID NO:4) isformed from the peptidyl-resin(Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH-Resin)(SEQ ID NO:3).

FIG. 4 shows a reaction scheme in which a TFA salt of AMG 416 is formedfrom the SPy intermediate (AA^(1-7(SPy))).

FIG. 5 shows a reaction scheme in which the HCl salt of AMG 416 isformed from the TFA salt of AMG 416.

FIG. 6 shows a reaction scheme in which Boc-D-Arg(Pbf)-OH is formed fromBoc-D-Arg-OH.

FIG. 7 shows a reaction scheme in which D-Arg(Pbf)-OH is formed fromBoc-D-Arg(Pbf)-OH.

DETAILED DESCRIPTION

The present disclosure now will be described more fully hereinafter.This disclosure may, however, be embodied in many different forms andshould not be construed as limited to the embodiments set forth herein;rather, these embodiments are provided so that this disclosure will bethorough and complete, and will fully convey its scope to those skilledin the art.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety,unless otherwise indicated. In an instance in which the same term isdefined both in a publication, patent, or patent applicationincorporated herein by reference and in the present disclosure, thedefinition in the present disclosure represents the controllingdefinition. For publications, patents, and patent applicationsreferenced for their description of a particular type of compound,peptide, chemistry, etc., portions pertaining to such compounds,chemistry, etc. are those portions of the document which areincorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures used in connection with, and techniques of,molecular biology and protein chemistry described herein are thosewell-known and commonly used in the art. The methods and techniques ofthe present application are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated. See,e.g., Laszlo, Peptide-Based Drug Design: Methods and Protocols, HumanaPress (2008); Benoiton, Chemistry of Peptide Synthesis, CRC Press(2005); Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992), which are incorporated herein by referencefor any purpose. Purification techniques are performed according tomanufacturer's specifications, as commonly accomplished in the art or asdescribed herein. The terminology used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art. Standardtechniques can be used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

It should be understood that this disclosure is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the disclosed, which is defined solely by the claims.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

I. General Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle, unless specifically indicated otherwise. By way of example, “anelement” means one element or more than one element.

The term “AMG 416”, also known as etelcalcetide, formerly known asvelcalcetide or KAI-4169, refers to a compound having the chemical name:N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-arginamidedisulfide with L-cysteine, which has the following structural formula:

Reference to AMG 416, or to any compound or AMG 416 fragment,intermediate, or precursor as described herein, is intended to encompassneutral, uncharged forms thereof, as well as pharmaceutically acceptablesalts, hydrates and solvates thereof.

The terms “AMG 416 hydrochloride” and “AMG 416 HCl” are interchangeableand refer to a hydrochloride salt form of AMG 416 having the followingstructural formula:

Generally, x has a value of 3-5 (e.g., 3, 4 or 5).

“Pharmaceutically acceptable salt” refers to a salt form of a compoundhaving at least one group suitable for salt formation that causes nosignificant adverse toxicological effects to a patient. The term“pharmaceutically-acceptable salt” may, in one respect, refer to therelatively non-toxic, inorganic or organic acid addition salts ofcompounds as provided herein, e.g., AMG 416, as well as AMG 416fragments, intermediates, precursors, and the like, possessing one ormore ionizable amine groups. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, andlaurylsulphonate salts and the like. (See, for example, Berge et al.(1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). Additionalsuitable pharmaceutically acceptable salt forms can be found in, e.g.,Handbook of Pharmaceutical Salts: Properties, Selection and Use,Weinheim/Zürich: Wiley-VCH/VHCA, 2002; P. H. Stahl and C. G. Wermuth,Eds.

As used herein, the terms “amino acid” and “residue” are interchangeableand, when used in the context of a peptide or polypeptide, refer to bothnaturally occurring and synthetic amino acids, as well as amino acidanalogs, amino acid mimetics and non-naturally occurring amino acidsthat are chemically similar to the naturally occurring amino acids. A“free amino acid” or “free amino group” refers to an amino acid, peptidefragment, or peptide having an amino group that is in the form of —NH₂,that is, is unprotected.

The phrase “protecting group” or “PG” as used herein refers to atemporary substituent or substituents that protect a potentiallyreactive functional group from an undesired chemical transformation.Examples of such protecting groups include esters of carboxylic acids,silyl ethers of alcohols, and acetals and ketals of aldehydes andketones, respectively. See, e.g., Greene, T. W.; Wuts, P. G. M.Protective Groups in Organic Synthesis, 4th ed.; Wiley: New York, 2007;Isidro-Llobet, A., et al., Amino Acid-Protecting Groups, Chem. Rev 2009,109, 2455-2504. Reactive amino acids or peptide fragments as describedherein often suitably contain one or more protecting groups onfunctionalities that are not the target of a subject chemicaltransformation. Exemplary protecting groups include, e.g.,carboxybenzyl, also referred to as benzyloxycarbonyl (“Cbz” or “Z”),9-fluorenylmethoxycarbonyl (Fmoc),2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf),tert-butyloxycarbonyl (Boc), trityl (Trt), methyl ester (OMe),2-pyridinesulfenyl (SPy or S-Pyr), amide, and the like. In the shorthandstructures provided herein, —NH₂ at the C-terminus signifies an amideprotecting group (˜C(O)NH₂), “H” at the N-terminus refers to a freeamino group, and designation of a protecting group in parenthesessignifies that the protecting group is on the δ nitrogen of ornithine.

As used herein, the term “protection eliminating agent” or “deprotectingagent” can be used interchangeably, and is a chemical reagent forremoving amino-protecting agents connected on amino acids, and theamino-protecting agent can be well-known in the field, such as, but notlimited to Fmoc and Boc.

As used herein, the terms “coupling agent”, “condensing agent”,“activating agent,” “condensation activating agent,” usedinterchangeably herein, refer to a chemical reagent that facilitatesreaction of an amino group from one amino acid with a carboxyl groupfrom another amino acid to form a peptide bond. Exemplary couplingagents are well-known in the art and include but are not limited tocarbodiimides such as N,N′-diisopropylcarbodiimide (DIC),dicyclohexylcarbodiimide (DCC),1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU),[benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium;tetrafluoroborate (TBTU),N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU), and N,N-diisopropylethyl amine (DIPEA). See,e.g., El-Faham, A. and Albericio, F., “Peptide Coupling Reagents, Morethan a Letter Soup”, Chem. Rev. 2011, 111, 6557-6602. Such compounds arereadily available from commercial vendors.

As used herein, the term “cleavage agent” refers to a chemical agentwhich can separate a peptide bound to a resin from the resin. Cleavageagents are well-known to those of ordinary skill in the art and includea acid solution comprising TFA and HCl solution.

The term “treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of signs or symptoms or making the injury, pathology orcondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; improving a patient's physical or mental well-being. Thetreatment or amelioration of signs or symptoms can be based on objectiveor subjective parameters; including the results of a physicalexamination. For example, certain methods presented herein successfullytreat SHPT in hemodialysis patients with CKD-MBD by decreasing serumintact parathyroid hormone (iPTH).

An “effective amount” is generally an amount sufficient to reduce theseverity and/or frequency of symptoms, eliminate the symptoms and/orunderlying cause, prevent the occurrence of symptoms and/or theirunderlying cause, and/or improve or remediate the damage that resultsfrom or is associated with the disease state (e.g., elevated PTHlevels). A “therapeutically effective amount” is an amount sufficient toremedy a disease state or symptoms, particularly a state or symptomsassociated with the disease state, or otherwise prevent, hinder, retardor reverse the progression of the disease state or any other undesirablesymptom associated with the disease in any way whatsoever. The fulltherapeutic effect does not necessarily occur by administration of onedose, and may occur only after administration of a series of doses.Thus, a therapeutically effective amount may be administered in one ormore administrations.

The terms “therapeutically effective dose” and “therapeuticallyeffective amount,” as used herein, means an amount that elicits abiological or medicinal response in a tissue system, animal, or humanbeing sought by a researcher, physician, or other clinician, whichincludes alleviation or amelioration of the signs or symptoms of thedisease or disorder being treated, i.e., an amount of velcalcetide thatsupports an observable level of one or more desired biological ormedicinal response, for example lowering iPTH.

The terms “peptide,” “polypeptide” and “protein” are interchangeable andrefer to a polymer of amino acid residues. The terms also apply to aminoacid polymers in which one or more amino acid residues is an analog ormimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers. The terms can also encompassamino acid polymers that have been modified, e.g., by the addition ofcarbohydrate residues to form glycoproteins, or phosphorylated.Peptides, polypeptides and proteins can be produced by a liquid-phasesynthesis or solid phase synthesis or by a genetically-engineered orrecombinant cell, and comprise molecules having the amino acid sequence.

A “variant” of a peptide or polypeptide comprises an amino acid sequencewherein one or more amino acid residues are inserted into, deleted fromand/or substituted into the amino acid sequence relative to anotherpolypeptide sequence. Variants include fusion proteins.

A “derivative” of a peptide or polypeptide is a peptide or polypeptidethat has been chemically modified in some manner distinct frominsertion, deletion, or substitution variants, e.g., via conjugation toanother chemical moiety. Such modification can include the covalentaddition of a group to the amino and/or carboxy termini of the peptideor polypeptide, e.g., acetylation of the amino terminus and/or amidationof the carboxy terminus of a peptide or polypeptide.

The term “amino acid” includes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. S.Golub and D. R. Green, eds.), Sinauer Associates: Sunderland, Mass.(1991), which is incorporated herein by reference for any purpose.Stereoisomers (e.g., D-amino acids) of the 19 conventional amino acids(except glycine), unnatural amino acids such as [alpha]-,[alpha]-disubstituted amino acids, N-alkyl amino acids, and otherunconventional amino acids may also be suitable components forpolypeptides and are included in the phrase “amino acid.” Examples ofunconventional amino acids include: homocysteine, ornithine,4-hydroxyproline, [gamma]-carboxyglutamate,[epsilon]-N,N,N-trimethyllysine, [epsilon]-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, [sigma]-N-methylarginine, and other similar amino acidsand imino acids (e.g., 4-hydroxyproline). In the polypeptide notationused herein, the left-hand direction is the amino terminal direction andthe right-hand direction is the carboxyl-terminal direction, inaccordance with standard usage and convention.

A “subject” or “patient” as used herein can be any mammal. In a typicalembodiment, the subject or patient is a human.

The term “q.s.” means adding a quantity sufficient to achieve a desiredfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

II. Embodiments

In one or more embodiments, AMG 416 hydrochloride is prepared via aseries of process stages as follows: exemplary stages include the solidphase peptide synthesis of a seven-member linear fragment (stage I) ofAMG 416, followed by cleavage of the peptide chain from the resin withconcomitant side chain deprotection and cysteine activation (stage II),followed by in-situ conjugation of the peptide chain with L-Cys(disulfide formation) to provide crude AMG 416 (stage III), followed, insome embodiments, immediately, by preparative HPLC and lyophilization toprovide purified AMG 416 TFA salt (stage IV). Stage IV is followed by asubsequent salt exchange (TFA to HCl) by precipitation, and in someembodiments, followed by microfiltration and lyophilization to providethe purified AMG 416 hydrochloride salt (stage V).

Solid Phase Peptide Synthesis

The seven-membered linear fragment of AMG 416 may be synthesized by anymethod known in the art, including solid phase peptide synthesis (SPPS).As used herein, the term “solid phase synthesis” or “solid phase peptidesynthesis” refers to a method, well-known to one of ordinary skill inthe art, in which a growing peptide chain is linked to a solid support.Solid phase synthesis typically comprises the steps of: (i) covalentlybinding a first amino acid (whose amino-group is blocked or “protected”)to a solid phase carrier; (ii) removing the protecting group from theamino-group using a deprotecting agent; (iii) activating the carboxyl ofa second amino acid (whose amino-group is blocked) and contacting thesecond amino acid with the first amino acid bound to the solid phasecarrier so that a dipeptide (whose amino-group is blocked) is obtained;(iv) repeating the peptide bond formation steps and thus the peptidechain is extended from C-terminal to N-terminal; and (v) removing theprotecting group of the amino-group and separating the peptide chainfrom the solid phase carrier with a cleavage agent to yield a peptide.

Suitable techniques of solid phase synthesis are well known in the art,and include those described in Merrifield, in Chem. Polypeptides, pp.335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem.Soc. 85:2149 (1963); Davis et al., Biochem. Intl. 10:394-414 (1985);Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. No.3,941,763; Finn et al., The Proteins, 3rd ed., vol. 2, pp. 105-253(1976); and Erickson et al., The Proteins, 3rd ed., vol. 2, pp. 257-527(1976). See, also Houben-Weyl, Methods of Organic Chemistry. AdditionalSupplementary Volumes to the 4^(th) Ed., Vol E22A, “Synthesis ofPeptides and Peptidomimetics”, Editor-in-Chief M. Goodman. Georg ThiemeVerlag: Stuttgard and New York. 2002, pp. 685-877; Chan, W. C., White,P. D., “Fmoc Solid Phase Peptide Synthesis, A Practical Approach”.Oxford University Press, (200), p. 9-109. Solid phase synthesis istypically a preferred technique of making individual peptides such asAMG 416, since it is often one of the most cost-effective methods ofmaking small peptides.

In some embodiments, the main chain linear fragment of AMG 416 isassembled using standard solid-phase peptide synthesis protocolsemploying Fmoc-protection strategy and, for example, a Rink amide (RAM)resin such as available from Sigma Aldrich, to provide the C-terminusresin-bonded amide, along with acetylation of the peptide N-terminus. Insome other embodiments, other resins and linkers may be used (e.g.,Ramage amide AM resin, also referred as tricyclic amide linker resin).In one embodiment, assembly of the main chain linear fragment comprisesthe steps of: (i) mixing an Fmoc-Rink amide AM resin with a deprotectingagent to obtain a Rink amide AM resin; (ii) condensingFmoc-D-Arg(Pbf)-OH with the Rink amide AM resin to obtain anFmoc-D-Arg(Pbf)-Rink amide AM resin; (iii) repeating the Fmocdeprotection in step (i) and the condensation between an amino acid anda polypeptide on the resin in step (ii) for each remaining amino acidresidue of the main chain linear fragment of AMG 416, proceeding fromthe C-terminal to the N-terminal (e.g., using Fmoc-D-Cys(Trt)-OH,Fmoc-D-Ala-OH and Fmoc-D-Arg(Pbf)-OH) to form a polypeptide resinrepresented by SEQ ID NO:2; and (iv) repeating the Fmoc deprotection instep (i) and acetylating the N-terminus to form a polypeptide resinrepresented by SEQ ID NO:3. See FIG. 2 .

(SEQ ID NO: 2) Fmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin] (SEQ ID NO: 3)Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin]

Typically, cleavage of the Fmoc protecting group is achieved using adeprotecting agent such as piperidine in DMF. In one embodiment,coupling of the Fmoc-protected amino acid is carried out in a solventsuch as dimethylformamide (DMF) using a suitable coupling agent such asthe carbodiimide coupling agent, N,N-diisopropylcarbodiimide (DIC),optionally in the presence of an additive such as ethyl2-cyano-2-(hydroxyimino) acetate (Oxyma) for all amino acids exceptcysteine. In the case of peptide chain elongation with cysteine,coupling is typically carried out using N,N-diisopropylcarbodiimide(DIC) in the presence of a benzotriazole additive such ashydroxybenzotriazole (HOBT) in a solvent system such as dimethylformamide-dichloromethane, (i.e., DMF, DCM, HOBt, DIC).

Acetylation of the N-terminus may be accomplished by any method known inthe art. In one embodiment, acetylation of the N-terminus is carried outusing, for example, acetic anhydride (Ac₂O) in pyridine and DMF.

Cleavage from Resin

The peptide may be separated from the support, and the protecting groupsmay be removed from the side chains by any means known in the art. See,e.g., Synthetic Peptides: A User's Guide (G. A. Grant, ed.), W.H.Freeman and Company, New York, 1992; and Chan, W. C., White, P. D. “FmocSolid Phase Peptide Synthesis, A Practical Approach”, Oxford UniversityPress (2000), p. 64-66 and 105-109.

In one embodiment, the peptidyl-resin is added to a cocktail solutioncomprising water (e.g., deionized water (DIW)) trifluoroacetic acid(TFA), triisopropylsilane (TIPS) and dipyridyldisulfide (DPDS). Thisallows the peptide to be separated from the resin with concomitantside-chain deprotection and cysteine activation, thus preparing for thein-situ conjugating to L-Cys. The seven-amino acid SPy intermediateproduct (AA^(1-7(SPy))) is produced. See FIG. 3 . The sequence of theSPy intermediate product is provided in SEQ ID NO:4.

(SEQ ID NO: 4) Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg- NH₂

In-Situ Conjugation and Preparative HPLC

The SPy intermediate product may be conjugated to L-Cys by any methodknown in the art. In one embodiment, conjugation of L-Cys is performedin aqueous TFA.

The AMG 416 (TFA salt) produced may be purified by any means known inthe art. In one embodiment, AMG 416 (TFA salt) is purified by highpressure liquid chromatography (HPLC). For example, in one embodiment,the purification and concentration of the AMG 416 (TFA salt) comprises areverse-phase HPLC purification step and a reverse-phase HPLCconcentration step. See FIG. 4 .

The purified and concentrated sample containing AMG 416 (TFA salt) maybe lyophilized.

Salt Conversion

The TFA salt may be converted to pharmaceutically acceptable salt, suchas the hydrochloride salt, by any means known in the art.

In one embodiment, the lyophilized TFA salt of AMG 416 is dissolved inan aqueous solution of isopropyl alcohol (IPA). The TFA salt solution isthen charged to an HCl solution for salt exchange and precipitation ofthe HCl salt. The precipitate may then be reconstituted with water,filtered thought a micro-filter (e.g., 0.2 μm filter) and lyophilized toisolate the HCl salt of AMG 416. See FIG. 5 .

Purification of a SPy Intermediate

In an alternative embodiment, the SPy intermediate product is purifiedprior to conjugation to the L-Cys. Generally, SPy intermediate products,in particular peptide-SPy intermediates, are considered to be highlyunstable, i.e., are thought to not be sufficiently stable to withstandefficient purification, such as by HPLC. However, in arriving at themethods provided herein, it was discovered by the Applicants that,unexpectedly, the peptide-SPy intermediates prepared according to themethods described herein are indeed sufficiently stable to withstand aseparate purification step. Moreover, it was further discovered thatpurification of such intermediates prior to conjugation to the L-Cys canactually increase the efficiency and decrease the cost of manufacture ofthe final peptide drug product.

In an exemplary embodiment, the alternative method is carried out asfollows. The method described below and in Example 5 is useful for thepurification of a SPy intermediate as provided herein, wherein theintermediate remains stable and is suitable for conjugation to athiol-containing moiety, e.g., via disulfide bond formation. Forexample, the peptide-SPy intermediate is dissolved in an aqueoussolution containing no more than about 0.2% TFA, for example, about0.05% to 0.15% TFA, or about 0.1% TFA, and is then directly applied toan HPLC column for chromatographic purification. Solvent exchange of theHPLC fractions containing the peptide-SPy intermediate can then becarried out, for example, by azeotropic distillation. Following solventexchange of the peptide-SPy intermediate into an appropriate solvent,such as a mixture of water-isopropanol, a thiol-containing moiety, suchas L-Cys is added directly to the peptide-SPy intermediate solution toeffect conjugation. The resultant conjugated product, e.g., in solution,is then available for salt exchange.

A particular embodiment of the foregoing purification method is asfollows. After the initial cleavage of the peptide from the resinsupport and isolation of the AA^(1-7(SPy)) intermediate product, theintermediate is dissolved in 0.1% TFA and acetonitrile, loaded onto astationary phase HPLC column and purified as described above. The use ofa 0.1% TFA solution has multiple advantages for HPLC purification whencompared to the use of, for example, a 0.2% TFA solution. For instance,0.1% TFA is less damaging to the stationary phase during thepurification process than a higher concentration TFA solution. That isto say, by using such an optimized concentration of TFA, there is lessdecomposition of the stationary phase, thereby resulting in a longerlifetime for the stationary phase. Moreover, the peptide-SPyintermediate (e.g., AA^(1-7(SPy))) product is less polar, resulting inthe intermediate being better retained on the reverse phase stationaryphase. As a result, much higher loading onto the stationary phase can beachieved in each purification run. In some embodiments, the increasedloading capacity increases the throughput of the manufacturing process1.5 to 2-fold, or about 1.5-fold.

The HPLC fractions containing the SPy intermediate product as a TFA saltare then subjected to azeotropic distillation with sufficient IPAcharges to change the solvent from the acetonitrile and water to a 15%water in IPA solution suitable for L-Cys conjugation and salt exchange.This method is particularly advantageous as both the conjugation andsalt exchange can be carried out in a single vessel, further improvingthe efficiency and feasibility of the manufacturing process.

Manufacture of Fmoc-D-Arg(Pbf)-OH Starting Material

AMG 416 comprises a linear sequence of 7 amino acids, 4 of which areD-arginines. Disclosed herein is a method for synthesizingFmoc-D-Arg(Pbf)-OH, the Fmoc derivative of D-arginine used in thesynthesis of AMG 416. Use of a high quality Fmoc-D-Arg(Pbf)-OH startingmaterial can furnish additional purity to crude AMG 416 from stage I tostage III, to thereby enhance the purification yield in stage IV. Moreimportantly, the use of a high quality, high purity starting materialcan provide a desirable and advantageous control element to secure thedesired purity of AMG 416. A new process for preparing high qualityD-Arg(Pbf)-OH and Fmoc-D-Arg(Pbf)-OH has been developed which produces ahigher yield, requires fewer unit operations, provides more robustquality control and is amenable to large scale manufacturing. Insummary, the method of preparing Fmoc-D-Arg(Pbf)-OH described hereinrepresents a significant increase in the feasibility of AMG 416manufacturing accompanied by a potential gain in quality.

A synthetic route that is one of the most concise syntheses ofFmoc-D-Arg(Pbf)-OH found in the literature and which can be used for thecommercial scale synthesis of Fmoc-D-Arg(Pbf)-OH is as follows. See,e.g., Chinese Patent No. CN101250172B, 2 May 2012. The synthesis startsby protecting the amino group of D-Arg with di-tert-butyl dicarbonate(Boc₂O) yielding Boc-D-Arg-OH in an approximately quantitative yieldafter the isolation. In step 2, the side chain guanidine group isprotected with a Pbf group in the presence of a base such as aqueoussodium hydroxide. The product is used directly in the next step as anIPA (isopropyl alcohol) solution without isolation. Step 3 comprisesremoving the Boc protecting group under acidic conditions, and isolatingthe corresponding product, D-Arg(Pbf)-OH, as a crystalline intermediate.Step 4 of the process comprises installing an Fmoc protecting group onthe amino functionality to afford Fmoc-D-Arg(Pbf)-OH as the finalproduct. Since both step 2 and step 4 products are amorphous materials,i.e., providing limited capability to reject impurities, the crystallineintermediate D-Arg(Pbf)-OH (step 3 product) serves as a control point toinfluence and secure the purity of the final product.

To obtain a high purity product is a significant challenge when relyingon processes reported in the literature. Multiple recrystallizations aretypically required to meet the purity requirement. As an example,D-Arg(Pbf)-OH is recrystallized seven times in an EtOH/EtOAc/watertertiary solvent system in order to upgrade the purity to a desiredlevel. In addition, in step 2, 20-30% of the starting materials remainunreacted even in the presence of a large excess of sodium hydroxide andPbfCl, and the overall yield (step 1 to 3) is typically only less than40%.

The improved process developed and described herein and illustrated inFIGS. 6 and 7 , provides several advantages over known processes. NaI isintroduced as a catalyst for converting Boc-D-Arg-OH toBoc-D-Arg(Pbf)-OH (see Step 2 in FIG. 6 ). The incorporation of sodiumiodide is effective to significantly improve the reaction kinetics andas a result, the conversion of step 2 can be increased to greater than95%, and the assay yield can be improved up to ˜90%. In addition, thetotal quantity of impurities is also reduced.

D-Orn(Pbf)-OH and the ethyl ester of D-Arg(Pbf)-OH are key impuritiesformed in step 2 and step 3 (FIG. 7 ), respectively, and are difficultto remove via crystallization, thereby contributing to the need formultiple recrystallizations. The improved process described hereinfurther comprises the use of isopropyl acetate (IPAc), a more stericallyhindered ester when compared to the more commonly used EtOAc, as thesolvent for step 3 in which Boc-D-Arg(Pbf)-OH is converted toD-Arg(Pbf)-OH. The use of IPAc significantly slows down the sidereaction, transesterification, catalyzed by the strong acid, HCl. As aresult, the content of corresponding impurity, the ester ofD-Arg(Pbf)-OH, is reduced to less than about 0.5% (vs greater than 1.0%in the process which employs EtOAc). Additionally, isopropyl alcohol(IPA)/water was found to be a more powerful solvent system which allowedremoval of all process impurities yet minimized product loss in thecrystallization steps. In a test run conducted at a laboratory scale (20g), the purity of D-Arg(Pbf)-OH (step 3 intermediate) was improved togreater than 99.7% with only two crystallizations, and the overall yield(step 1 to 3) was approximately 70%. One embodiment of this improvedprocess is described in Example 6 below.

The skilled artisan will readily appreciate that the present disclosurealso extends to variants and derivatives of AMG 416. For example, in oneembodiment, the methods provided herein may also be used withN-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-arginamidedisulfide with D-cysteine. In another embodiment, the disclosedformulations also may be used withN-acetyl-L-cysteinyl-L-alanyl-L-arginyl-L-arginyl-L-arginyl-L-alanyl-L-arginamidedisulfide with D-cysteine and/orN-acetyl-L-cysteinyl-L-alanyl-L-arginyl-L-arginyl-L-arginyl-L-alanyl-L-arginamidedisulfide with L-cysteine. In another embodiment, the disclosedformulation may also be used withN-acetyl-D-cysteinyl-D-arginyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-arginamidedisulfide with L-cysteine, and/orN-acetyl-D-cysteinyl-D-arginyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-arginamidedisulfide with D-cysteine. Additionally, the instant methods may beemployed to prepare one or more of the compounds provided in Table 1,Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9and/or Table 10 of International Pat. Publication No. WO 2011/014707. Infurther embodiments, the methods described herein may also be used toprepare compounds described in International Pat. Publication No. WO2011/014707.

EXAMPLES

The following examples, including the experiments conducted and theresults achieved, are provided for illustrative purposes only and arenot to be construed as limiting the scope of the appended claims.

Example 1A Synthesis of the Main Chain Linear Fragment

Synthesis of the main chain linear fragment is shown in FIG. 2 . RinkAmide AM Resin (Fmoc 2,4-dimethoxy-4′(carboxymethyloxy)-benzhydrylaminelinked to Aminomethyl Resin) (1 kg) was added to DMF (5.8 L/kg) and thesolution was agitated at 22° C. The resin was filtered and slurrywashed. A sample was tested for residual Fmoc (UV test) and residualpiperidine (pH). The Kaiser and/or TNBS color tests were conducted toensure that Fmoc deprotection had been carried out.

The first six amino acid derivative couplings followed the sameprocedure for pre-activation, coupling and washing. 1.6 eq of theprotected Fmoc amino acid was added to DMF, 8.9 L/kg at 22° C. Oxyma(2.45 eq) was then added. The solution was cooled to 21° C. and DIC(2.13 eq) was added, and the reaction was allowed to proceed. Thepre-activated solution was combined with resin and the reaction wasallowed to proceed. DIC (1.07 eq) was added and the reaction was allowedto proceed at 22° C. A sample was tested for incomplete coupling usingthe Kaiser and/or TNBS color tests. The material was washed, followed byFmoc deprotection and further washing. A sample was tested for residualFmoc (UV test) and residual piperidine (pH). The Kaiser and/or TNBScolor tests were conducted to ensure Fmoc deprotection had taken place.

Fmoc-D-Cys(Trt)-OH 1.6 eq was added to a 1:1.7 DMF:DCM solution, 12L/kg, followed by addition of HOBt.H₂O (2.45 eq). The solution wascooled to 20° C. and DIC (2.13 eq) was added and the reaction wasallowed to proceed. The Pre-activated solution was combined with resinand the reaction was allowed to proceed at 22° C. DIC 1.07 eq wascharged to the SPPS reaction, where the DMF/DCM ratio was about 1:1. Thereaction was allowed to proceed at 22° C. A sample was tested forincomplete coupling using the Kaiser and/or TNBS color tests.

The material was washed followed by Fmoc deprotection and furtherwashing. A sample was tested for residual Fmoc (UV test) and residualpiperidine (pH). The Kaiser and/or TNBS color tests were conducted toensure Fmoc deprotection had taken place.

DMF (0 L/kg); Acetic Anhydride (1.06 L/kg) and Pyridine (1.06 L/kg) wereadded to the solution and the solution was agitated for pre-activation.The pre-activation solution was combined with the resin and agitated at22° C. The material was filtered and washed. A sample was tested forincomplete capping using the Kaiser and/or TNBS color tests. Thematerial was slurry washed. The resin was dried under nitrogen withoutagitation. A sample of the dry resin was taken and tested for LOD andresidual solvent. See FIG. 2 .

Example 1B Synthesis of the Main Chain Linear Fragment

Synthesis of the main chain linear fragment of AMG 416 is shown in FIG.2 . The peptide chain was built-up from the C-terminus to the N-terminuson Rink AM amide resin, 1 amino acid per cycle. Each cycle consists of 2reaction steps: 1) Fmoc cleavage from the N-terminus; 2) Coupling of thenext Fmoc-protected amino acid or final acetylation.

Start of SPPS: Rink AM amide resin (1.0 mole) was transferred into aSPPS reactor and washed with N,N′-Dimethylformamide (DMF).

Fmoc cleavage: The resin from the previous step was suspended in asolution of 20% piperidine in DMF for at least 10 min. Completion ofFmoc cleavage was monitored by ultraviolet (UV) absorption measurements.After completion of Fmoc cleavage, the resin was washed alternately withDMF and isopropanol (IPA) until a neutral pH was achieved.

Fmoc-amino acids coupling: Following the Fmoc cleavage step, couplingreaction was performed by mixing the resin with the solution ofFmoc-protected amino acid derivative (>1.2 mole) and activating reagents(>1.8 moles) (N,N′-diisopropylcarbodiimide (DIC) and ethyl(hydroxyimino)-cyanoacetate (Oxyma)) in DMF. For coupling ofFmoc-D-Cys(Trt)-OH, DIC and 1-hydroxybenzotriazole hydrate (HOBt) wereused as activating agents, and a mixture of DMF and dichloromethane(DCM) was employed as the reaction solvent. The reaction mixture wasstirred at ambient temperature overnight. Kaiser and TNBS tests wereperformed to monitor the completion of the coupling. Negative resultsfrom both Kaiser and TNBS tests were required before the process wasmoved on to the next cycle. After each coupling or capping step, theresin was alternately washed with DMF and IPA.

Final acetylation: After the final Fmoc-deprotection, the N-terminalamino group of the peptide was acetylated using acetic anhydride andpyridine in DMF. Kaiser and TNBS tests were performed to check thecompletion of the acetylation. If the acetylation was incomplete, thesame acetylation procedure was then repeated until negative results fromboth Kaiser and TNBS tests were obtained.

Finally, the protected peptide backbone of the drug substance on theresin (AMG 416-Resin) was isolated by filtration, washed with DMF, IPAand acetonitrile (ACN) and dried under reduced pressure.

Example 2A Cleavage of Main Chain Linear Fragment from Resin

A cocktail solution was prepared by combining DIW (0.16 L/kg); TFA (5.64L/kg); TIPS (0.46 eq), and DPDS, (6.41 eq) at room temperature and thencooling the solution to 0±2° C. The peptide on the resin was added tothe cocktail solution at 0±2° C. and the solution was heated to 25° C.and the reaction was allowed to proceed. The resin was removed byfiltration and washed. The solution was held at −10° C. and a 6.8:1solution of IPE:MeCN (24.5 L/kg) at −10° C. was added over time tocontrol the temperature and precipitation. The reaction was allowed toproceed and the AMG 416 SPy intermediate product was filtered at −5° C.and washed. The SPy intermediate product was dried at 20° C. under fullvacuum. See FIG. 3 .

Example 2B Cleavage of Main Chain Linear Fragment from Resin

The cleavage solution was prepared in a reactor by mixing TFA, H₂O, andtriisopropylsilane (TIPS) in an approximate ratio of 96.9:2.6:0.5(v/v/v). To the cleavage solution, DPDS (>1.2 moles) was added as theactivating reagent of the sulfhydryl group of D-cysteine. AMG 416-Resin(1.0 mole) was charged to the reactor and the reaction mixture wasstirred for >1 h at room temperature. The resin was filtered off. Thefiltrate and washing solutions were transferred into another reactor andcooled. A cold anti-solvent mixture of diisopropyl ether (IPE) and ACNwas then charged to the solution to precipitate AMG 416-SPy. Thesuspension was filtered through a filter-drier and the filter cake ofAMG 416-SPy was subsequently washed with ACN and IPE and dried atapproximately 20° C. under reduced pressure on the filter drier.

Example 3 In-Situ Conjugation to L-Cys/Preparative HPLC

The AMG 416 SPy intermediate (1.0 mole) was added to a 0.2% TFAsolution. L-Cysteine (>1.1 moles) was added to the solution and thereaction was allowed to proceed at room temperature for at least 15 min.

The purification of the crude AMG 416 was carried out by preparativechromatography using a C18 silica gel stationary phase using ACN/H₂O asthe mobile phase and TFA as the modifier. The crude AMG 416 solutionfrom Stage III was loaded on the column and a linear gradient method wasused for the purification step. Elution was monitored by UV absorbanceat 230 nm. After each loading, the column was flushed with 80% ACN inwater (v/v) until a stable UV baseline was achieved. The fractions werestored at 5° C., they were sampled and then fractions having a desiredpurity (determined by HPLC) were pooled. The combined pools from thepurification run were concentrated by performing the concentration runusing the same HPLC column. The fractions were stored at 5° C. Thefractions with desired purity (determined by HPLC) were lyophilized toisolate the AMG 416 TFA salt. See FIG. 4 .

Example 4 Salt Conversion

The AMG 416 TFA salt was added to a solution of 15% water in IPA (v/v)10 L/kg at 10° C. until full dissolution was observed. The solution wasadded to a solution of 12M aqueous HCl, 0.27 L/kg and IPA 49.4 L/kg over3 hours via subsurface addition, resulting in direct precipitation ofthe AMG 416 4.5 HCl salt. The batch was aged for 3 hours and sampled foranalysis.

The material was filtered and slurry washed with 96 wt % IPA, 10 L/kg.The cake was then re-slurried for 4 hours in 10 L/kg of 96% wt % IPA.The material was filtered and further slurry washed with 96% IPA, 10L/kg and then IPA 10 L/kg. The material was dried under full vacuum at25° C. The dry cake was dissolved in water 8 L/kg and the batch isconcentrated via distillation to remove residual IPA and achieve thedesired concentration. The solution temperature was kept below 25° C.throughout the distillation. See FIG. 5 .

Example 5 Purification of the SPy Intermediate and Production of AMG 416HCl

An alternative method for preparation of AMG 416 HCl salt is describedhere. As described in Example 2 above, the SPy intermediate product wasdried at 20° C. under full vacuum after cleavage from the resin,precipitation and filtration. The precipitate was then dissolved in a0.1% TFA aqueous solution and loaded onto a C-18 column for HPLCpurification. The column was run at <60 bar and the solution temperaturewas 15-25° C. throughout. The eluents were 0.1% TFA in acetonitrile and0.1% TFA in water. The fractions were stored at 5° C., they were sampledand then fractions were pooled. The combined pools from two runs werediluted and a concentration/purification run was performed using thesame HPLC column to decrease the total volume and remove additionalimpurities. The fractions were stored at 5° C.

The fractions containing the AMG 416 SPy intermediate were subjected toazeotropic distillation to change the solvent from the 0.1% TFA to a 15%water in IPA solution, charging with IPA as needed. To the resultant AMG416 SPy intermediate in IPA solution was then added L-Cysteine 1.15 eqand the reaction was allowed to proceed at room temperature forconjugation to occur and to form the AMG 416 TFA salt as described abovein Example 4. The AMG 416 TFA solution was added to a solution of 12Maqueous HCl, 0.27 L/kg and IPA 49.4 L/kg over 3 hours via subsurfaceaddition, resulting in direct precipitation of the AMG 416 4.5 HCl salt.The batch was aged for 3 hours and sampled for analysis.

The material was filtered and slurry washed with 96 wt % IPA, 10 L/kg.The cake was then re-slurried for 4 hours in 10 L/kg of 96% wt % IPA.The material was filtered and further slurry washed with 96% IPA, 10L/kg and then IPA 10 L/kg. The material was dried under full vacuum at25° C. The dry cake was dissolved in water 8 L/kg and the batch wasconcentrated via distillation to remove residual IPA and achieve thedesired concentration. The solution temperature was kept below 25° C.throughout the distillation.

Example 6 Synthesis of H-D-Arg(Pbf)-OH

Synthesis of the main chain linear fragment as described in Example 1requires the use of Fmoc-D-Arg(Pbf)-OH for addition of the D-argininesubunits, which make up 4 of the 7 residues in the linear fragment. Animproved and more efficient method for synthesizing Fmoc-D-Arg(Pbf)-OHis described below.

The synthesis starts with the protection of the amino group of D-Arg byDi-tert-butyl dicarbonate (Boc₂O), yielding Boc-D-Arg-OH inapproximately quantitative yield after isolation by crystallizationunder the standard procedure reported in the literature. The arginineside chain guanidine group was protected with a Pbf group by a one-houraddition (0-5° C.) of a 10/1 acetone/THF solution of Pbf-Cl (1.3 eq) inthe presence of aqueous NaOH (4.3 eq)/NaI (5% mol) as the base,producing Boc-D-Arg(Pbf)-OH (85-90% assay yield) See FIG. 6 . TheBoc-D-Arg(Pbf)-OH in IPAc solution was treated with 4.8 eq ofconcentrated HCl at 20° C. for approximately 6 h. After the reaction,the organic layer was discarded and the crude product was isolated fromthe aqueous layer by adjusting the pH to 5 using NaOH, after which awhite suspension was observed. See FIG. 7 . When the supernatant had aconcentration of approximately 3 mg/mL at 20° C., the supernatant wasfiltered at room temperature, and the resultant cake washed with waterand dried under a vacuum. The overall assay yield after this step wasabout 80-85%. The purity of H-D-Arg(Pbf)-OH was increased to >98.5% by afirst re-crystallization from 3/1 water/IPA (v/v). When the supernatanthad a concentration of about 7 mg/mL it was filtered and dried undervacuum. After this step, the overall assay yield was about 75%.(Typically about 10% of the product loss is observed in this step). Asecond re-crystallization step from 4/1 water/IPA (v/v) was performed.Filtering was done when the supernatant concentration was about 3.7mg/mL. The second re-crystallization increased the purity ofH-D-Arg(Pbf)-OH to about 99.84 area percentage purity (LCAP) by HPLCwith no impurities more than 0.2 LCAP. The typical yield of this step isabout 93% with about 5% product loss. The H-D-Arg(Pbf)-OH was thenreacted with FmocOSu according to standard protocols and the productFmoc-D-Arg(Pbf)-OH was isolated using the standard procedure in theliterature.

What is claimed is:
 1. A method for preparing etelcalcetide or apharmaceutically acceptable salt thereof, comprising: providing apeptide having the structure ofAc-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂ (SEQ ID NO:4); andcontacting the peptide with L-Cys to produce a conjugated product. 2.The method of claim 1, wherein said contacting comprises dissolving thepeptide in an aqueous solution comprising L-Cys and trifluoroacetic acid(TFA).
 3. The method of claim 2, wherein said contacting occurs at roomtemperature for about 15 minutes.
 4. The method of claim 1, furthercomprising lyophilizing the conjugated product.
 5. The method of claim4, further comprising contacting the conjugated product with an aqueoussolution comprising isopropyl alcohol and hydrochloride (HCl), therebyproducing a precipitate comprising etelcalcetide HCl (SEQ ID NO:1). 6.The method of claim 5, further comprising washing the precipitatecomprising etelcalcetide HCl (SEQ ID NO:1) with isopropyl alcohol. 7.The method of claim 6, further comprising dissolving the isopropylalcohol washed precipitate in water to produce a dissolved precipitate.8. The method of claim 7, further comprising concentrating the dissolvedprecipitate via distillation.
 9. The method of claim 5, furthercomprising purifying the precipitate by HPLC.
 10. The method of claim 1,wherein the conjugated product is represented by a formulaAc-c(C)rrrar-NH₂ (SEQ ID NO: 1) or a pharmaceutically acceptable saltthereof.
 11. The method of claim 10, further comprising contacting theconjugated product with an aqueous solution comprising isopropyl alcohol(IPA) and HCl, thereby producing a precipitate a HCl salt of SEQ IDNO:
 1. 12. The method of claim 4, wherein the method further comprisesderiving the peptide having structureAc-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂ (SEQ ID NO:4) fromproviding a resin-bound peptide having a structure ofAc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[Resin](SEQ ID NO:3).
 13. The method of claim 12, wherein the derivingcomprises contacting the resin-bound peptide with a solution comprisingwater, trifluoroacetic acid, triisopropylsilane and dipyridyldisulfide.14. The method of claim 1, wherein the peptide comprises a TFA salt ofAc-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH₂ (SEQ ID NO:4).